Probe for measuring phytase activity

ABSTRACT

A myo-inositol derivative: 
     
       
         
         
             
             
         
       
     
     R 1 , R 2 , R 3  and R 4  are identical, being selected from PO 3 H 2 , PO 3 Na 2 , PO 3 K 2 , PO 3 Li 2 , PO 3 Ca, PO 3 Mg, PO 3 (NH 4 ) 2 , PO 3 (RNH 3 ) 2 , PO 3 (R 2 NH 2 ) 2 , PO 3 (R 4 N) 2 , PO(OR) 2 , H, COZ and BHPP, where R is benzyl or alkyl of 1 to 6 carbon atoms and Z is an alkyl or arylalky group providing a cleavable protecting group; R 5  is one of H, benzyl, 4-methoxybenzyl, COZ, PO 3 H 2 , PO 3 Na 2 , PO 3 K 2 , PO 3 Li 2 , PO 3 Ca, PO 3 Mg, PO 3 (NH 4 ) 2 , PO 3 (RNH 3 ) 2 , PO 3 (R 2 NH 2 ) 2 , PO 3 (R 4 N) 2 , PO(OR) 2 , and BHPP, where R is benzyl or alkyl of 1 to 6 carbon atoms and Z is an alkyl or arylalky group providing a cleavable protecting group; X is one of CH 2 , CH 2 CH 2 O, and CH 2 CH 2 CH 2 O; n is an integer from 1 to 8; L 1  is a single bond or CH 2 ; L 2  is one of a single bond, CONH, CH 2 CONH, CH 2 CH 2 CONH, CH 2 CH 2 NHCO, CH 2 CH 2 NHCONH, CH 2 CH 2 NHCSNH, CH 2 CH 2 NHSO 2 , CH 2 CH 2 CH 2 NHCO, CH 2 CH 2 CH 2 NHCONH, CH 2 CH 2 CH 2 NHCSNH, CH 2 CH 2 CH 2 NHSO 2 , NHCO, NHCONH, NHCSNH, OCONH, CH 2 , CH 2 CH 2 , CH 2 CH 2 O, CH 2 CH 2 S, CH 2 CH 2 NH, CH 2 CH 2 CH 2 , CH 2 CH 2 CH 2 O, CH 2 CH 2 CH 2 S, CH 2 CH 2 CH 2 NH, and NH—SO 2 ; and R 6  is a UV-visible chromophore, a UV-chromophore, a fluorescent moiety, or a radiolabeled moiety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of, and claims the benefit ofpriority from, U.S. patent application Ser. No. 11/249,231, filed Oct.13, 2005, which application claims the benefit of priority from U.S.Provisional Patent Application Ser. No. 60/674,950, filed Apr. 26, 2005.The entire contents of the foregoing cases are incorporated herein byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not applicable.

FIELD OF THE INVENTION

The present invention pertains to probes for measuring phytase activity,and more particularly to such probes comprising a myo-inositolderivative compound of the following nominal formula:

BACKGROUND

The majority of phosphorous (P) in farm animal feed grains is present asa mixed salt of myo-inositol hexakisphosphate, shown below, and morecommonly referred to as phytic acid (“myo-IP₆”).

Because grain-consuming animals such as swine and poultry do not producethe enzyme phytase, myo-IP₆ is largely unavailable as aphosphorus-containing nutrient source and is thus excreted in highconcentrations in the waste of these animals. Such waste is, however,applied to croplands as a means of enhancing soil fertility, thoughlittle information exists regarding the process of how myo-IP₆ istransformed into a crop-available nutrient. At least in part, this lackof information respecting the fate of myo-IP₆ in soil and water-sedimentenvironments is attributable to the absence of artificial substratesthat can be used for the convenient measurement of phytase activity.Still, recent experimental evidence suggests that bacterial phytaseplays an important role in the (bio)chemical transformation.

Phytases catalyze the sequential hydrolysis of myo-IP₆, formingorthophosphate (ortho-P) and a series of partially dephosphorylatedphosphoric esters of myo-inositol. In some cases, hydrolysis may go tocompletion yielding the parent compound myo-inositol. Based onbiochemical properties of the amino acid sequence alignment, other havecharacterized phytases into two major classes, the histidine acidphytases (comprising the PhyA, PhyB, and PhyC groups), to which most ofthe bacterial and fungal phytases belong, and the alkaline phytases(PhyD). The phytate-degrading enzyme, 3-phytase (myo-inositolhexakisphosphate 3-phosphohydrolase, EC 3.1.3.8; PhyA and PhyB groups)hydrolyzes myo-IP₆ preferentially at the C-3 position, while 6-phytase(myo-inositol hexakisphsophate 6-phosphohydrolase, EC 3.1.3.26; PhyC)hydrolyzes myo-IP₆ preferentially at the C-6 position.

Conventional phytase assays conducted on bacterial cell wall-free lysateor whole cell lysate routinely call for the addition of myo-IP₆ tobuffered cell lysate and subsequent measurement of the released ortho-Pby colorimetric analysis. However, experiments by the inventor hereofdemonstrates substantial ortho-P release from bacterial cell lysateresulting from “cell free” phosphate-mediated hydrolysis ofcell-associated phosphate compounds, including accumulated intracellularpolyphosphate. Notably, bacteria, archaea and fungi are all capable ofproducing polyphosphate. Thus, it would seem that the conventionalortho-P release assay is not a specific measure of phytase activity inlysed cell preparations, and its use under such conditions may result inan exaggerated estimate of phytase activity or, possibly, even a falsepositive test result.

In light of the foregoing, it would be desirable to provide a specificand sensitive quantitative enzyme assay capable of measuring phytaseactivity in cell culture filtrates, cell-lysate preparations, soils,etc.

SUMMARY OF THE INVENTION

The specification discloses a myo-inositol derivative compoundcharacterized by the following nominal formula:

Wherein R¹, R², R³ and R⁴ are identical and are selected from the groupconsisting of PO₃H₂, PO₃Na₂, PO₃K₂, PO₃Li₂, PO₃Ca, PO₃Mg, PO₃(NH₄)₂,PO₃(RNH₃)₂, PO₃(R₂NH₂)₂, PO₃(R₄N)₂, PO(OR)₂, H, COZ and BHPP, where R isbenzyl or alkyl of 1 to 6 carbon atoms and Z is an alkyl or arylalkygroup that provides a cleavable ester protecting group; R⁵ is selectedfrom the group consisting of H, benzyl, 4-methoxybenzyl, COZ, PO₃H₂,PO₃Na₂, PO₃K₂, PO₃Li₂, PO₃Ca, PO₃Mg, PO₃(NH₄)₂, PO₃(RNH₃)₂, PO₃(R₂NH₂)₂,PO₃(R₄N)₂, PO(OR)₂, and BHPP, where R is benzyl or alkyl of 1 to 6carbon atoms and Z is an alkyl or arylalky group that provides acleavable ester protecting group; X is selected from the groupconsisting of CH₂, CH₂CH₂O, and CH₂CH₂CH₂O; n is an integer from 1 to 8;L¹ is selected from the group consisting of a single bond and CH₂; L² isselected from the group consisting of a single bond, CONH, CH₂CONH,CH₂CH₂CONH, CH₂CH₂NHCO, CH₂CH₂NHCONH, CH₂CH₂NHCSNH, CH₂CH₂NHSO₂,CH₂CH₂CH₂NHCO, CH₂CH₂CH₂NHCONH, CH₂CH₂CH₂NHCSNH, CH₂CH₂CH₂NHSO₂, NHCO,NHCONH, NHCSNH, OCONH, CH₂, CH₂CH₂, CH₂CH₂O, CH₂CH₂S, CH₂CH₂NH,CH₂CH₂CH₂, CH₂CH₂CH₂O, CH₂CH₂CH₂S, CH₂CH₂CH₂NH, and NH—SO₂; and R⁶ isselected from the group consisting of UV-visible chromophores,UV-chromophores, fluorescent moieties, and radiolabeled moieties.

According to another embodiment of this derivative compound, R¹, R², R³,R⁴ and R⁵ are selected from the group consisting of PO₃H₂, PO₃Na₂,PO₃(NH₄)₂, and BHPP, X is CH₂ or CH₂CH₂O, n is an integer from 2 to 8,L¹ is a single bond or is CH₂, L² is selected from the group consistingof a single bond, CONH, NHCO, NHCONH, NHCSNH, OCONH, NHSO₂, CH₂CH₂CONH,CH₂CH₂CH₂NH, CH₂CH₂CH₂NHCO, CH₂CH₂CH₂NHCONH, CH₂CH₂CH₂NHCSNH,CH₂CH₂CH₂OCONH, and CH₂CH₂CH₂NHSO₂, and R⁶ is selected from the groupconsisting of UV-visible chromophores, UV-chromophores, fluorescentmoieties, and radiolabeled moieties. In one variation, R⁶ is selectedfrom the group consisting of UV-chromophores and fluorescent moieties.In a further variation of this embodiment, X is CH₂, n is 6, and L² isselected from the group consisting of NH—CO and NH—CS—NH.

According to another embodiment, R¹, R², R³, R⁴ and R⁵ are selected fromthe group consisting of PO₃H₂, PO₃Na₂, PO₃ (NH₄)₂, and BHPP, X is CH₂, nis 6, L¹ is a single bond or is CH₂, L² is selected from the groupconsisting of NHCO and NH—CS—NH, R⁶ is selected from the groupconsisting of UV-chromophores and fluorescent moieties. In a furthervariation of this embodiment, L² is NHCO and R⁶ is phenyl. In stillanother variation, R¹, R², R³, R⁴ and R⁵ are all PO₃(NH₄)₂, PO₃H₂, or H.

According to yet another embodiment, R¹, R², R³, R⁴ and R⁵ are selectedfrom the group consisting of PO₃H₂, PO₃Na₂, PO₃(NH₄)₂, and BHPP, X isCH₂, n is 6, L¹ is a single bond or is CH₂, L² is NHCO or NHCSNH, and R⁶is fluorescein-5-yl. In further variations of this embodiment, R¹, R²,R³, R⁴ and R⁵ are all PO₃(NH₄)₂, PO₃H₂, or H.

According to still another embodiment, R¹, R², R³, R⁴ and R⁵ areselected from the group consisting of PO₃H₂, PO₃Na₂, PO₃(NH₄)₂, andBHPP, X is CH₂, n is 6, L¹ is a single bond or is CH₂, L² is NHCO orNHCSNH, and R⁶ is fluorescein-6-yl. In further variations of thisembodiment, R¹, R², R³, R⁴ and R⁵ are all PO₃(NH₄)₂, PO₃H₂, or H.

According to a further embodiment, R¹, R², R³, R⁴ and R⁵ are selectedfrom the group consisting of PO₃H₂, PO₃Na₂, PO₃(NH₄)₂, and BHPP,CH₂CH₂O, n is 3 or 4, L¹ is a single bond or is CH₂, L² is a single bondor CH₂CH₂CH₂NH, and R⁶ is 7-nitro-2-oxa-1,3-diazol-4-yl. In furthervariations of this embodiment, R¹, R², R³, R⁴ and R⁵ are all PO₃(NH₄)₂,PO₃H₂, or H.

Per one form of this embodiment, R⁵ is selected from the groupconsisting of H, benzyl, and 4-methoxybenzyl; X is CH₂; n is an integerfrom 2 to 8; L¹ is a single bond or is CH₂, L² is selected from thegroup consisting of CO—NH, NH—CO, NH—CO—NH, NH—CS—NH, O—CO—NH, andNH—SO₂; and R⁶ is a UV-chromophore or a fluorescent moiety.

In another form, L is NH—CO and R⁶ is phenyl. Alternatively, L isNH—CS—NH and R⁶ is fluorescein-5-yl. Per still another form, L isNH—CS—NH and R⁶ is fluorescein-6-yl.

In another embodiment, R¹, R², R³, R⁴ and R⁵ are all PO₃(NH₄)₂, X isCH₂, n is 5, L¹ is CH₂, and L² is NHCO. R⁶ is selected from the groupconsisting of UV-chromophores and fluorescent moieties. In one form ofthis embodiment, R⁶ is phenyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the synthesis of an exemplary phytic acidprobe (compound 2) according to the instant invention;

FIG. 2 comprises a series of HPLC chromatograms depicting the sequentialdephosphorylation of the phytic acid probe of FIG. 1 by 3-phytase;

FIGS. 3 a through 3 c show the kinetics of ortho-phosphate release upon3-phytase-catalysed dephosphorylation of the phytic acid probe of FIG. 1(FIG. 3 a), the accumulation and eventual decline of the triphosphateproduct, indicating the rate-limiting step (FIG. 3 b), and a progresscurve showing the release of ortho-phosphate upon dephosphorylation ofthe phytic acid probe of FIG. 1 (FIG. 3 c);

FIG. 4 shows HPLC chromatograms illustrating the appearance ofT-myo-inositol (compound 7 of FIG. 1) from the completedephosphorylation of the phytic acid probe of FIG. 1; and

FIG. 5 shows a series of HPLC chromatograms depicting the sequentialdephosphorylation of the phytic acid probe of FIG. 1 by 6-phytase.

WRITTEN DESCRIPTION

The following definitions are applicable in this written specification:

“Alkane”: A linear, branched, or cyclic compound containing hydrogen andcarbon connected by single bonds.

“Alkyl”: A linear, branched, or cyclic moiety containing hydrogen andcarbon connected by single bonds.

“Aryl”: A cyclic moiety containing at least one six-carbon ring withthree double bonds.

“Benzyl”: The chemical moiety that is phenylmethyl.

“BHPP”: (Bis(1,2-hydroxymethyl)phenyl)phosphate, the cyclic phosphateester moiety that contains the1,5-dihydrobenzo[e][2-oxo-1,3,2-dioxaphosphepan]-2-yl moiety.

“Benzyl acetal”: The reaction product of a 1,2-diol with benzaldehydethat includes the structural moiety of 2-phenylmethyl-1,3-dioxolane.

“Chromophore”: A chemical moiety which absorbs selected wavelengths oflight.

“Cyclohexane ketal”: The reaction product of a 1,2-diol withcyclohexanone that includes the structural moiety of2-(spirocyclohexyl)-1,3-dioxolane.

“Cyclopentane ketal”: The reaction product of a 1,2-diol withcyclopentanone that includes the structural moiety of2-(spirocyclopentyl)-1,3-dioxolane.

“Diethyl ketal”: The reaction product of a 1,2-diol with 3-pentanonethat includes the structural moiety of 2,2-diethyl-1,3-dioxolane.

“Dimethyl ketal”: The reaction product of a 1,2-diol with acetone thatincludes the structural moiety of 2,2-dimethyl-1,3-dioxolane.

“Fluorescein”: A fluorescent compound of the formula C₂₀H₁₂O₅.

“Heteroaryl”: An aromatic moiety containing at least one five-memberedring with two double bonds or at least one six-membered ring with threedouble bonds, either of which contains one or more heteroatoms.

“Heteroatom”: An atom that is O, N or S.

“4-Methoxybenzyl acetal”: The reaction product of a 1,2-diol with4-methoxybenaldehyde that includes the structural moiety of2-(4-methoxybenzyl)-1,3-dioxolane.

“Pentacyclic acetal”: The reaction product of a 1,2-diol with analdehyde that includes the structural moiety of the five-membered ringthat is 1,3-dioxolane.

“Pentacyclic ketal”: The reaction product of a 1,2-diol with a ketonethat includes the structural moiety of the five-membered ring that is1,3-dioxolane.

“Phenyl”: A cyclic moiety of the formula C₆H₅.

“UV-chromophore”: A chemical moiety that absorbs selected wavelengths ofultraviolet light. Exemplary UV-chromophores include, withoutlimitation, aryl and heteroaryl moieties.

“UV-visible-chromophore”: A chemical moiety that absorbs selectedwavelengths of ultraviolet and visible light.

Referring now to the written specification and the drawings, the presentinvention will be seen to most generally comprise a myo-inositolderivative compound characterized by the following nominal formula:

Wherein R¹, R², R³ and R⁴ are identical, and are selected from the groupconsisting of PO₃H₂, PO₃Na₂, PO₃K₂, PO₃Li₂, PO₃Ca, PO₃Mg, PO₃(NH₄)₂,PO₃(RNH₃)₂, PO₃(R₂NH₂)₂, PO₃(R₄N)₂, PO(OR)₂, H, COZ and BHPP, where R isbenzyl or alkyl of 1 to 6 carbon atoms and Z is an alkyl or arylalkygroup that provides a cleavable ester protecting group; R⁵ is selectedfrom the group consisting of H, benzyl, 4-methoxybenzyl, COZ, PO₃H₂,PO₃Na₂, PO₃K₂, PO₃Li₂, PO₃Ca, PO₃Mg, PO₃(NH₄)₂, PO₃(RNH₃)₂, PO₃(R₂NH₂)₂,PO₃(R₄N)₂, PO(OR)₂, and BHPP, where R is benzyl or alkyl of 1 to 6carbon atoms and Z is an alkyl or arylalky group that provides acleavable ester protecting group; X is selected from the groupconsisting of CH₂, CH₂CH₂O, and CH₂CH₂CH₂O; n is an integer from 1 to 8;L¹ is selected from the group consisting of a single bond and CH₂; L² isselected from the group consisting of a single bond, CONH, CH₂CONH,CH₂CH₂CONH, CH₂CH₂NHCO, CH₂CH₂NHCONH, CH₂CH₂NHCSNH, CH₂CH₂NHSO₂,CH₂CH₂CH₂NHCO, CH₂CH₂CH₂NHCONH, CH₂CH₂CH₂NHCSNH, CH₂CH₂CH₂NHSO₂, NHCO,NHCONH, NHCSNH, OCONH, CH₂, CH₂CH₂, CH₂CH₂O, CH₂CH₂S, CH₂CH₂NH,CH₂CH₂CH₂, CH₂CH₂CH₂O, CH₂CH₂CH₂S, CH₂CH₂CH₂NH, and NH—SO₂; and R⁶ isselected from the group consisting of UV-visible chromophores,UV-chromophores, fluorescent moieties, and radiolabeled moieties.

The cleavable ester protecting groups comprehend, without limitation,those protecting groups which can be cleaved by treatment with a lipase,according to known techniques.

According to another embodiment of this derivative compound, R¹, R², R³,R⁴ and R⁵ are selected from the group consisting of PO₃H₂, PO₃Na₂,PO₃(NH₄)₂, and BHPP, X is CH₂ or CH₂CH₂O, n is an integer from 2 to 8,L¹ is a single bond or is CH₂, L² is selected from the group consistingof a single bond, CONH, NHCO, NHCONH, NHCSNH, OCONH, NHSO₂, CH₂CH₂CONH,CH₂CH₂CH₂NH, CH₂CH₂CH₂NHCO, CH₂CH₂CH₂NHCONH, CH₂CH₂CH₂NHCSNH,CH₂CH₂CH₂OCONH, and CH₂CH₂CH₂NHSO₂, and R⁶ is selected from the groupconsisting of UV-visible chromophores, UV-chromophores, fluorescentmoieties, and radiolabeled moieties. In one variation, R⁶ is selectedfrom the group consisting of UV-chromophores and fluorescent moieties.In a further variation of this embodiment, X is CH₂, n is 6, and L² isselected from the group consisting of NH—CO and NH—CS—NH.

According to another embodiment, R¹, R², R³, R⁴ and R⁵ are selected fromthe group consisting of PO₃H₂, PO₃Na₂, PO₃ (NH₄)₂, and BHPP, X is CH₂, nis 6, L¹ is a single bond or is CH₂, L² is selected from the groupconsisting of NHCO and NH—CS—NH, R⁶ is selected from the groupconsisting of UV-chromophores. and fluorescent moieties. In a furthervariation of this embodiment, L² is NHCO and R⁶ is phenyl. In stillanother variation, R¹, R², R³, R⁴ and R⁵ are all PO₃(NH₄)₂, PO₃H₂, or H.

According to yet another embodiment, R¹, R², R³, R⁴ and R⁵ are selectedfrom the group consisting of PO₃H₂, PO₃Na₂, PO₃(NH₄)₂, and BHPP, X isCH₂, n is 6, L¹ is a single bond or is CH₂, L² is NHCO or NHCSNH, and R⁶is fluorescein-5-yl. In further variations of this embodiment, R¹, R²,R³, R⁴ and R⁵ are all PO₃(NH₄)₂, PO₃H₂, or H.

According to still another embodiment, R¹, R², R³, R⁴ and R⁵ areselected from the group consisting of PO₃H₂, PO₃Na₂, PO₃(NH₄)₂, andBHPP, X is CH₂, n is 6, L¹ is a single bond or is CH₂, L² is NHCO orNHCSNH, and R⁶ is fluorescein-6-yl. In further variations of thisembodiment, R¹, R², R³, R⁴ and R⁵ are all PO₃(NH₄)₂, PO₃H₂, or H.

According to a further embodiment, R¹, R², R³, R⁴ and R⁵ are selectedfrom the group consisting of PO₃H₂, PO₃Na₂, PO₃(NH₄)₂, and BHPP,CH₂CH₂O, n is 3 or 4, L¹ is a single bond or is CH₂, L² is a single bondor CH₂CH₂CH₂NH, and R⁶ is 7-nitro-2-oxa-1,3-diazol-4-yl. In furthervariations of this embodiment, R¹, R², R³, R⁴ and R⁵ are all PO₃(NH₄)₂,PO₃H₂, or H.

Per one form of this embodiment, R⁵ is selected from the groupconsisting of H, benzyl, and 4-methoxybenzyl; X is CH₂; n is an integerfrom 2 to 8; L¹ is a single bond or is CH₂, L² is selected from thegroup consisting of CO—NH, NH—CO, NH—CO—NH, NH—CS—NH, O—CO—NH, andNH—SO₂; and R⁶ is a UV-chromophore or a fluorescent moiety.

In another form, L is NH—CO and R⁶ is phenyl. Alternatively, L isNH—CS—NH and R⁶ is fluorescein-5-yl. Per still another form, L isNH—CS—NH and R⁶ is fluorescein-6-yl.

In another embodiment, R¹, R², R³, R⁴ and R⁵ are all PO₃(NH₄)₂, X isCH₂, n is 5, L¹ is CH₂, and L² is NHCO. R⁶ is selected from the groupconsisting of UV-chromophores and fluorescent moieties. In one form ofthis embodiment, R⁶ is phenyl.

The inventive compounds as contemplated by the present invention includestereoisomers.

EXPERIMENTAL EXAMPLE 1 Derivation of an Exemplary Probe

Referring now to FIG. 1, derivation of an exemplary compound—namely,5-O-[6-(benzoylamino)hexyl]-D-myo-inositol-1,2,3,4,6-pentakisphosphate(2) according to the present invention will be better understood.

The compound 4-O-benzyl-1,6:2,3-di-O-cyclohexylidine-myo-inositol 3 wasselected as the starting material since it allows for ether-linkedderivatization at the 5-position (phytic acid numbering), it positionsthe linker “meta” to the initial site of reaction (i.e., the3-position), thus minimizing the chances that the linker (pluschromophore) would interfere with the preferential active site of a3-phytase, and further simplifies the stereochemical issues viaplacement of the linker in the meso plane. An ester linkage, bycontrast, would be less desirable as being hydrolytically stable andmore resistant to phosphatase activity.

Following the procedure of Garegg et al., as published in CarbohydrateRes. 1984, 130, 322, the inventors hereof prepared 100 g of the pureisomer 3 of FIG. 1 with flash chromatography followed byrecrystallization. The protected myo-inositol 3 was alkylated at theopen 5-position using the mesylate of 6-azidohexa-1-ol and sodiumhydride in DMF. It was found that moderate heat was necessary to ensurea complete reaction. More specifically, 2.8 grams (70 mmol) of 60% NaH(mineral oil suspension) was added to a solution comprising 15 grams(34.7 mmol) of the compound 3 in 190 ml DMF. The mixture was stirred for45 minutes and then 1-azzodihexyl 6-O-mesylate (19.2 grams, 86.8 mmol)was added dropwise over a period of 15 minutes. The mixture wasthereafter stirred for a further 5 minutes before placing the reactionflask in an oil bath preheated to 60° C. After 18 hours incubation thereaction was checked by thin-layer chromatography (“tlc”) (Hexanes-EtOAc(4:1), visualization with H₂SO₄/EtOH/hot plate) which indicated thecomplete absence of the compound 3 and the presence of a faster movingproduct. The reaction mixture was cooled to room temperature and pouredinto a separatory funnel containing 1.4 L EtOAc. The mixture was washedfour times with 400 ml portions of water, once with 300 ml of brine, andthe aqueous washes discarded. The organic layer was subsequently dried(Na₂SO₄), decanted from the drying agent and concentrated in vacuo toafford the crude resinous product. Subsequent purification was achievedusing flash silica gel (400 g) column purification. The column waspacked in Hexanes-EtOAc 20:1, and loaded with the crude product(dissolved in 30 ml of the Hexanes-EtOAc 20:1 mixtures). The column waseluted first with the Hexanes-EtOAc 20:1 (2.1 L), then with 17.5:1 (1.85L) and finally with 15:1 (1.6 L), collecting 125 ml fractions. Pureproduct was contained in fractions 20-44. These fractions were combined,concentrated in vacuo (aspirator vacuum, 35° C.) to afford a syrup whichwas co-evaporated from 250 ml of anhydrous dichloromethane. The productwas then dried at 0.1 torr at 45° C. for 4 hours to give 17.3 g (89.5%yield).

Simple hydrogenation of the compound 4 over 10% Pd/C reduced thesidechain azide to an amine with concomitant removal of the ringO-benzyl group affording a 55% yield of the product 5. Morespecifically, a 2 L hydrogenation vessel was charged with 600 ml of a5:1 mixture of THF:MeOH, 16.5 g (29.7 mmol) of the compound 4, purgedwith Argon, and 14 g of 10% Pd/C added. The reaction vessel was thenhydrogenated on a Parr shaker at 40 PSI for 4 days. The Pd/C wasfiltered off and the filtrate concentrated to afford 13.45 g of thecompound 5 as a resin. To remove traces of MeOH from this product, itwas co-evaporated two times from 50 ml portions of anhydrous DCM.

Selective benzoylation of the amino group in 5 was accomplished withbenzoyl cyanide in dichloromethane giving a 77% yield of the product 6.Specifically, to a solution comprising the compound 5 (10.2 g, 23.2mmol) in 160 ml anhydrous DCM was added, dropwise with stirring via anaddition funnel, a solution of benzoyl cyanide (3.36 g, 25.63 mmol) in160 ml of anhydrous DCM. The reaction mixture was stirred for 16 hours,and thereafter 75 ml of saturated aqueous NaHCO₃ added, and the mixturestirred for a further 45 minutes. The aqueous wash was discarded and theorganic layer dried over Na₂SO₄. The drying agent was removed byfiltration, and the filtrate concentrated to afford a resin.Purification was achieved using flash silica gel (440 g) columnpurification. The column was packed in Hexanes-EtOAc 3:2, and loadedwith the crude product (dissolved in 20 ml of the Hexanes-EtOAc 1:1mixture). The column was eluted with the Hexanes-EtOAc (3:2, 1.5 L),collecting 75 ml fractions. Pure product was contained in fractions21-33. These fractions were combined, concentrated in vacuo (aspiratorvacuum, 35° C.) to afford a solid which twice was dissolved in 50 ml ofanhydrous dichloromethane, and re-concentrated, dried under vacuum (0.1torr) at room temperature, to afford 7.4 g (58.6%) of the compound 6.

When compound 6 was subsequently heated in an acetic acid/water mixtureat 100° C. for 45 minutes, the 1,6:2,3 cyclohexylidene groups wereselectively removed, giving the 5-O-derivitized myo-inositol 7 in 94%yield. More particularly, to a mixture of acetic acid (32 mL) and water(8 mL) was added 3.0 g (5.52 mmol) of the compound 6 and the reactionmixture warmed at 100° C. for 1 hour whereby tlc (Hexanes-EtOAc 1:1)indicated the absence of starting material. The mixture was cooled toroom temperature and the solvents removed under 0.1 torr vacuum on arotary evaporator at ≦35° C. to afford a white solid. This material wasco-evaporated four times from 100 mL portions of toluene, and theresultant solid slurried with ethyl ether and collected by filtration.The product was dried in an aberhalden in vacuo (0.1 torr) at 78° C.over P₂O₅ for 16 hours to give 2.0 g (94%) of the compound 7 as a whitesolid, mp 232-236° C.

Through the reaction of the compound 7 withN,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine in thepresence of tetrazole, followed by oxidation with m-CPBA, the compound 8was prepared in 88% yield. More particularly, 1.15 g (16.41 mmol, 10.5equivalents) tetrazole was added to a stirred solution of the compound 7(600 mg, 1.56 mmol) in 40 mL of anhydrous MeCN to yield a lightsuspension. A solution ofN,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine (2.6 g, 10.87mmol, 7 equivalents) in 20 mL of anhydrous acetonitrile was addeddropwise with stirring to this suspension. The reaction mixture wasstirred for 36 hours. Tlc on silica gel plates (Hexanes-Ethyl Acetate,(3:2)), showed a major non-polar product at R_(f)=0.62, along with baseline impurities. When the tlc plate was developed in DCM/MeOH (50:1),the “base line” spot yielded 6-8 faint but distinguishable components,along with the major product (now with an R_(f) near the solvent front).At this point, 27 ml of 0.5 M iodine in a THF/Pyridine/H₂O (10:5:1) wasadded. The “iodine tinted” mixture was stirred for 2 hours at roomtemperature. Tlc of the reaction mixture showed that the major non-polarproduct with R_(f)=0.62 (elution with Hexanes-Ethyl Acetate (3:2)) to beno longer present, with only a base line spot. Tlc of the reactionmixture in DCM/MeOH (20:1) now showed a major spot of R_(f)=0.67, alongwith some faint baseline impurities. The reaction was deemed complete,and poured into 600 ml of EtOAc. The mixture was washed once with 200 mlof 10% aqueous sodium bisulfite, once with 200 ml of saturated aqueousbicarbonate and finally with 100 ml of brine. The organic layer wasdried over anhydrous sodium sulfate, filtered, and the resultingfiltrate concentrated under aspirator vacuum at 35° C. to afford a solidresidue. This residue was slurried with 20 ml of acetone, and filteredto collect an off-white solid, 440 mg. Tlc, on silica gel, of this solidproduct revealed it as the major product seen at R_(f)=0.67 in thereaction mixture. This solid was subsequently dissolved in 25 ml ofMeOH, and adsorbed in to 5 g of powdered Na₂SO₄. The solid wasthereafter placed onto a flash silica gel (20 g) column packed inDCM/MeOH (20:1), and eluted, initially with the same for a total of 200ml, and then switching to 10:1, collecting 20 ml fractions. Thefractions 3, 4 and 5, containing pure product with the R_(f)=0.67, werecombined and concentrated in vacuo to afford a white solid, 150 mg, at149-152° C. solid transforms to a “glass,” then at 250° C. (dec).

³¹P NMR of the compound 8 showed three singlets in a 2:1:2 ratio,consistent with a molecule containing a meso plane and equilibration ofchair conformations. Removal of the protecting groups from compound 8was accomplished by hydrogenation over 5% Pd/C, giving compound 2 as apenta-dihydrogen phosphate which was then directly, without isolation,converted to the decaammonium salt 2 via treatment with ammoniumhydroxide. The overall yield for these last two steps was 96%. Morespecifically, the compound 8 (140 mg, 0.108 mmol) was added to 20 ml ofMeOH, the mixture warmed slightly to dissolve, and 3 ml of THF and 4 mlof H₂O added. The reaction vessel was purged with N₂, and 200 mg of 5%Pd/C added. The reaction vessel was placed on a Parr shaker and purgedwith NH₂ several times, and thereafter placed under a head pressure of50 PSI H₂. The mixture was hydrogenated for 16 hours at roomtemperature, following which 1.1 mmol of ammonium hydroxide was added.The mixture was subsequently filtered through celite to remove thecatalysts, and the resulting filtrate was concentrated in vacuo (1 torr,35° C.) to afford a colorless, opaque glass. The crude product was thenre-dissolved in distilled water and filtered through a 0.2 micron TEFLONmembrane filter, and the filtrate then re-concentrated to give a whitesolid. This solid product was slurried in Et₂O, and filtered to give thecompound 2, which compound was then dried under 0.1 torr at roomtemperature to yield 99 mg (96%) of the final product 2.

³¹P NMR for compound 2 consisted of three singlets at 0.56, 1.19 and3.12 ppm (integration 2:1:2), consistent with the phosphates at C-1 andC-3 being equivalent, as with the phosphates at C-4 and C-6, and withthe singlet at 1.19 ppm (integration one) attributable to the C-2 mesoplane phosphate group. The ¹H NMR spectrum confirmed the structuralcomponents, i.e., linker, benzamido and ring methines, for structure 2.Further definitive evidence for 2 was obtained from the mass spectrum.Though a negative ion Maldi mass spectrum did give an [M-H]-ion at m/z782, determination of an exact mass was not possible. However, the exactmass of 782.0168 (predicted 782.0183), consistent with the desiredproduct 2 anion, was achieved by negative ion electrospray.

EXPERIMENTAL EXAMPLE II 3- and 6-Phytase Assays

Referring now to FIGS. 2 through 5, the aforesaid exemplary probeaccording to the instant invention (compound 2 of FIG. 1) was employedin assays of 3-phytase and 6-phytase activity. The 3-phytase assayconsisted of a solution combining A. ficuum 3-phytase (0.27 U) and thepenta-ammonium salt of compound 2 (1.2 mM in glycine-HCl buffer 0.2M, pH2.6, 3 mL). The 3-phytase assay was carried out at 37° C. The 6-phytaseassay consisted of a solution combining wheat 6-phytase (0.27 U) andcompound 2 (1.2 mM in sodium acetate buffer, 0.2M, pH 5.2, 3 mL). The6-phytase assay was carried out at 50° C.

Control assays were similarly prepared but lacked the phytase enzyme.Assays were carried out in tinted vials, capped with septa and stirredcontinuously. Aliquot samples (30 μL) were taken and diluted withmethanol-water (58:42, 970 μL) for HPLC analysis over the 24-hour timecourse of the assay.

Mobile phase (pH 4.0) was prepared by mixing methanol (580 mL), water(420 mL), tetrabutylammonium hydroxide (8 mL), 5N aqueous sulfuric acid(0.5 mL), and 6.4 mM aqueous phytic acid (0.2 mL). HPLC analysis wasperformed on an analytical HPLC system using a reversed phase column(Hamilton PRP-1, 5 μm, 150×4.6 mm). Compounds were quantified by theexternal standards method.

Typical assay output for 3-phytase is shown in FIG. 2 wherein thedisappearance of compound 2 (peak d) and the sequential appearance anddecline of the analogous tetraphosphate (peak c), the triphosphate (peakb), and the monophosphate (peak a) are observed with increasingincubation time. The concomitant release of ortho-phosphate may also bemonitored for compound 2 (FIG. 3 a) and myo-IP₆ (FIG. 3 c). Theaccumulation of the triphosphate (peak b in FIG. 2 and FIG. 3 b) and thelack of a significant peak for the diphosphate indicate that the ratelimiting step of the production of compound 7 from compound 2 isconversion of triphosphate to diphosphate. After 24 hours of incubation,compound 2 is completely converted to compound 7 in the presence of3-phytase (FIG. 4). In contrast, no conversion was seen in the enzymefree control. Neither in the assay nor in the control is there anyevidence of amide bond cleavage that would separate the UV chromophorefrom the phytic acid core. In other words, no benzoic acid could bedetected by HPLC. Analogous assay output for 6-phytase is shown in FIG.5, which demonstrates that compound 2 serves equally well as a substratefor 6-phytase.

It will be appreciated from the above disclosure that the presentinvention improves upon the prior art by providing probes which act asoptically detectable substrates in a specific, sensitive, andquantitative assay of phytase enzyme activity.

Of course, the foregoing is merely illustrative of the presentinvention, and those of ordinary skill in the art will appreciate thatmany additions and modifications to the present invention, as set out inthis disclosure, are possible without departing from the spirit andbroader aspects of this invention as defined in the appended claims.

All publications identified above are hereby incorporated herein byreference in their entireties.

1. A myo-inositol derivative compound of the following nominal formula:

Wherein R¹, R², R³ and R⁴ are identical, and are selected from the groupconsisting of PO₃H₂, PO₃Na₂, PO₃K₂, PO₃Li₂, PO₃Ca, PO₃Mg, PO₃(NH₄)₂,PO₃(RNH₃)₂, PO₃(R₂NH₂)₂, PO₃(R₄N)₂, PO(OR)₂, H, COZ and BHPP, where R isbenzyl or alkyl of 1 to 6 carbon atoms and Z is an alkyl or arylalkygroup that provides a cleavable ester protecting group; R⁵ is selectedfrom the group consisting of H, benzyl, 4-methoxybenzyl, COZ, PO₃H₂,PO₃Na₂, PO₃K₂, PO₃Li₂, PO₃Ca, PO₃Mg, PO₃(NH₄)₂, PO₃(RNH₃)₂, PO₃(R₂NH₂)₂,PO₃(R₄N)₂, PO(OR)₂, and BHPP, where R is benzyl or alkyl of 1 to 6carbon atoms and Z is an alkyl or arylalky group that provides acleavable ester protecting group; X is selected from the groupconsisting of CH₂, CH₂CH₂O, and CH₂CH₂CH₂O; n is an integer from 1 to 8;L¹ is a single bond or CH₂; L² is selected from the group consisting ofa single bond, CONH, CH₂CONH, CH₂CH₂CONH, CH₂CH₂NHCO, CH₂CH₂NHCONH,CH₂CH₂NHCSNH, CH₂CH₂NHSO₂, CH₂CH₂CH₂NHCO, CH₂CH₂CH₂NHCONH,CH₂CH₂CH₂NHCSNH, CH₂CH₂CH₂NHSO₂, NHCO, NHCONH, NHCSNH, OCONH, CH₂,CH₂CH₂, CH₂CH₂O, CH₂CH₂S, CH₂CH₂NH, CH₂CH₂CH₂, CH₂CH₂CH₂O, CH₂CH₂CH₂S,CH₂CH₂CH₂NH, and NH—SO₂; and R⁶ is selected from the group consisting ofUV-visible chromophores, UV-chromophores, fluorescent moieties, andradiolabeled moieties.
 2. The compound of claim 1, wherein R¹, R², R³,R⁴ and R⁵ are selected from the group consisting of PO₃H₂, PO₃Na₂,PO₃(NH₄)₂, and BHPP, X is CH₂ or CH₂CH₂O, n is an integer from 2 to 8,L² is selected from the group consisting of a single bond, CONH, NHCO,NHCONH, NHCSNH, OCONH, NHSO₂, CH₂CH₂CONH, CH₂CH₂CH₂NH, CH₂CH₂CH₂NHCO,CH₂CH₂CH₂NHCONH, CH₂CH₂CH₂NHCSNH, CH₂CH₂CH₂OCONH, and CH₂CH₂CH₂NHSO₂. 3.The compound of claim 2, wherein R⁶ is selected from the groupconsisting of UV-chromophores and fluorescent moieties.
 4. The compoundaccording to claim 2, X is CH₂, n is 6, L¹ is a single bond, and L² isselected from the group consisting of NHCO and NHCSNH.
 5. The compoundof claim 4, wherein L² is NHCO and R⁶ is phenyl.
 6. The compound ofclaim 5, wherein R¹, R², R³, R⁴ and R⁵ are all PO₃(NH₄)₂.
 7. Thecompound of claim 5, wherein R¹, R², R³, R⁴ and R⁵ are all PO₃H₂.
 8. Thecompound of claim 5, wherein R¹, R², R³, R⁴ and R⁵ are all H.
 9. Thecompound of claim 2, wherein X is CH₂, n is 6, L¹ is a single bond, L²is NHCO or NHCSNH, and R⁶ is fluorescein-5-yl.
 10. The compound of claim2, wherein X is CH₂, n is 6, L¹ is a single bond, L² is NHCO or NHCSNH,and R⁶ is fluorescein-6-yl.
 11. The compound of claim s 9 or 10, whereinR¹, R², R³, R⁴ and R⁵ are PO₃(NH₄)₂.
 12. The compound of either ofclaims 9 or 10, wherein R¹, R², R³, R⁴ and R⁵ are PO₃H₂.
 13. Thecompound of claim 9 or 10, wherein R¹, R², R³, R⁴ and R⁵ are all H. 14.The compound of claim 2, wherein X is CH₂CH₂O, n is 3 or 4, L² is asingle bond or CH₂CH₂CH₂NH, and R⁶ is 7-nitro-2-oxa-1,3-diazol-4-yl. 15.The compound of claim 14, wherein R¹, R², R³, R⁴ and R⁵ are PO₃(NH₄)₂.16. The compound of claim 14, wherein R¹, R², R³, R⁴ and R⁵ are PO₃H₂.17. The compound of claim 14, wherein R¹, R², R³, R⁴ and R⁵ are H. 18.The compound of claim 1, wherein R¹, R², R³, R⁴ and R⁵ are allPO₃(NH₄)₂, X is CH₂, n is 5, L¹ is CH₂, and L² is NHCO.
 19. The compoundof claim 18, wherein R⁶ is phenyl.